Location of the Zn2+-Binding Site on S100B As Determined by NMR Spectroscopy and Site-Directed Mutagenesis

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Abstract
In addition to binding Ca2+, the S100 protein S100B binds Zn2+ with relatively high affinity as confirmed using isothermal titration calorimetry (ITC; Kd = 94 ± 17 nM). The Zn2+-binding site on Ca2+-bound S100B was examined further using NMR spectroscopy and site-directed mutagenesis. Specifically, ITC measurements of S100B mutants (helix 1, H15A and H25A; helix 4, C84A, H85A, and H90A) were found to bind Zn2+ with lower affinity than wild-type S100B (from 2- to >25-fold). Thus, His-15, His-25, Cys-84, His-85, and perhaps His-90 of S100B are involved in coordinating Zn2+, which was confirmed by NMR spectroscopy. Previous studies indicate that the binding of Zn2+ enhances calcium and target protein-binding affinities, which may contribute to its biological function. Thus, chemical shift perturbations observed here for residues in both EF-hand domains of S100B during Zn2+ titrations could be detecting structural changes in the Ca2+-binding domains of S100B that are pertinent to its increase in Ca2+-binding affinity in the presence of Zn2+. Furthermore, Zn2+ binding causes helix 4 to extend by one full turn when compared to Ca2+-bound S100B. This change in secondary structure likely contributes to the increased binding affinity that S100B has for target peptides (i.e., TRTK peptide) in the presence of Zn2+.